Detection of secreted polypeptides

ABSTRACT

The invention relates to methods of detecting a secreted polypeptide produced by a cell, as well as to methods for selecting a cell that produces high levels of the secreted polypeptide. Such methods can be used to select a cell producing high levels of a secreted polypeptide encoded by a heterologous nucleic acid that has been introduced into the cell.

TECHNICAL FIELD

This invention relates to methods of detecting secreted polypeptides,and more particularly to methods for selecting cells that produce highlevels of secreted polypeptides.

BACKGROUND

Secreted proteins generally contain at their amino terminus a signalsequence that directs the ribosomes synthesizing them to the endoplasmicreticulum ER). Protein synthesis is completed on ribosomes attached tothe rough ER membrane. Completed polypeptide chains move to the Golgicomplex and subsequently are sorted to various destinations. Proteinssynthesized and sorted in the secretory pathway include not only thosethat are secreted from the cell, but also proteins resident in the lumenof the ER, Golgi, and lysosomes, as well as integral proteins in themembranes of these organelles and the plasma membrane.

Most newly made proteins in the ER are incorporated into small transportvesicles that either fuse with the cis-Golgi or with each other to formmembrane stacks known as the cis-Golgi reticulum. From the cis-Golgi,certain proteins are retrieved to the ER via a different set ofretrograde transport vesicles. In the process called cisternal migrationa new cis-Golgi stack with its cargo of luminal protein physically movesfrom the cis position (nearest the ER) to the trans position (farthestfrom the ER), successively becoming first a medial-Golgi cisterna andthen a trans-Golgi cisterna. During this process, membrane and luminalproteins are constantly retrieved from later to earlier Golgi cisternaeby small retrograde transport vesicles. By this process, enzymes andother Golgi resident proteins become localized either in the cis- ormedial- or trans-Golgi cisternae.

Proteins destined to be secreted from a cell move by cisternal migrationto the trans face of the Golgi and then into a complex network ofvesicles termed the trans-Golgi reticulum. From there, a secretoryprotein is sorted into a secretory vesicle. In all cell types, at leastsome of the secretory proteins are secreted continuously. These proteinsare sorted in the trans-Golgi network into transport vesicles thatimmediately move to and fuse with the plasma membrane, releasing theircontents by exocytosis. In some cells, the secretion of a specific setof proteins is not continuous. These proteins are sorted in thetrans-Golgi network into secretory vesicles that are stored inside thecell awaiting a stimulus, e.g., the binding of a hormone to itsreceptor, for exocytosis.

SUMMARY

The invention is based, at least in part, on the discovery that asecreted polypeptide can be detected on the surface of a cell thatproduces the polypeptide. The detection of a secreted polypeptide on thesurface of a cell can be used as a marker for cellular productivity ofthe secreted polypeptide. Accordingly, such methods can be used toselect a cell producing high levels of a given secreted polypeptide.

In one aspect the invention features a method of selecting a cellproducing a secreted polypeptide, the method including: providing a cellpopulation, wherein the cell population contains a cell containing aheterologous nucleic acid encoding a secreted polypeptide; contactingthe cell population with a compound that specifically binds to thesecreted polypeptide; detecting the binding of the compound to thesecreted polypeptide on the surface of the cell; and selecting the cellbased upon the presence or amount of the compound bound to the secretedpolypeptide on the surface of the cell.

In another aspect, the invention features a method of generating a cellproducing a secreted polypeptide, the method including: introducing intoa cell a heterologous nucleic acid encoding a secreted polypeptide;culturing the cell under conditions that allow for synthesis of thesecreted polypeptide; contacting the cell with a compound thatspecifically binds to the secreted polypeptide; detecting expression ofthe secreted polypeptide by binding of the compound to the secretedpolypeptide on the surface of the cell; and selecting the cell byfluorescence activated cell sorting.

A “secreted polypeptide” refers to a protein that is synthesized andsorted in the secretory pathway of a cell and is subsequently releasedfrom the cell in a soluble form. A “secreted polypeptide” typicallycontains an amino terminus signal sequence that is cleaved prior to therelease of the polypeptide from the cell. A “secreted polypeptide” doesnot refer to a species of a protein that exists as an integral membraneprotein or that is released from a cell by the cleavage of an integralmembrane protein, e.g., wherein the cleavage event releases a solubleextracellular region of the integral membrane protein.

“Selecting a cell” refers to a process of assigning a cell to a givenphysical location. In the context of the present invention, a cell isassigned a physical location based upon the presence or amount of acompound bound to a secreted polypeptide on the surface of the cell.Cells not having the desired characteristic are typically not assignedto the same physical location as a selected cell. The phrase “selectinga cell” includes, for example, depositing a cell (optionally togetherwith other cells having the same or similar characteristics) in acollection vessel based upon fluorescence properties of the cell asidentified by flow cytometry. Other examples of methods for selecting acell include magnetic separation and panning techniques.

A “heterologous nucleic acid” refers to a nucleotide sequence that hasbeen introduced into a cell by the use of recombinant techniques.Accordingly, a “heterologous nucleic acid” present in a given cell doesnot naturally occur in the cell (e.g., has no corresponding identicalsequence in the genome of the cell) and/or is present in the cell at alocation different than that where a corresponding identical sequencenaturally exists (e.g., the nucleotide sequence is present in adifferent location in the genome of the cell or is present in the cellas a construct not integrated in the genome). A “heterologous nucleicacid” does not refer to a nucleotide sequence that is present in a cellas a result of a cell fusion event between two or more cells.

The cell can be, for example, a eukaryotic cell (e.g., a mammalian cellsuch as a Chinese Hamster Ovary (CHO) cell or a COS cell) or aprokaryotic cell. The cell can be derived from a cell line or can be aprimary cell. In one embodiment, the cell is not a transformed cell. Inanother embodiment, the cell is not a B cell or a cell formed by fusionof a B cell and another cell.

The secreted polypeptide can be an antibody, e.g., a humanized antibody.

The compound can be labeled, e.g., fluorescently labeled. The compoundcan be an antibody, e.g., a fluorescently labeled antibody.

In one embodiment, the binding of the antibody to the secretedpolypeptide on the surface of the cell is detected by flow cytometry.The cell can optionally be selected by fluorescence activated cellsorting.

The cell can be selected together with a plurality of cells in the cellpopulation displaying the compound bound to the secreted polypeptide onthe surface of the plurality of cells. The plurality of cells canoptionally contain, e.g., at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, ormore of the cells in the cell population. The plurality of cells canoptionally contain no more than, e.g., at least 1%, 5%, 10%, 20%, 30%,40%, or 50% of the cells in the cell population.

The cell can be deposited in a vessel containing no cells in addition tothe cell.

A method described herein can further include culturing the selectedcell to produce a second cell population that produces the secretedpolypeptide; contacting the second cell population with the antibody;detecting the binding of the antibody to the secreted polypeptide on thesurface of a cell in the second cell population; and selecting the cellin the second cell population by fluorescence activated cell sortingbased upon the presence or amount of the antibody bound to the secretedpolypeptide on the surface of the cell. In one embodiment, thecontacting of the cell population with the antibody is carried outbetween 4° C. and 10° C., e.g., at about 4° C.

A method described herein can further include culturing the selectedcell in culture medium under conditions that allow for secretion of thesecreted polypeptide into the culture medium; and purifying the secretedpolypeptide from the culture medium.

In another aspect, the invention features a method of determining thepresence or amount of a secreted polypeptide produced by a cell, themethod including: contacting a cell producing a secreted polypeptidewith a compound that specifically binds to the secreted polypeptide,wherein the cell is not a B cell or a cell formed by the fusion of a Bcell with another cell; and detecting the binding of the compound to thesecreted polypeptide on the surface of the cell, to thereby determinethe presence or amount of the secreted polypeptide produced by the cell.

In one embodiment, the cell contains a heterologous nucleic acidencoding the secreted polypeptide.

The cell can be, for example, a eukaryotic cell (e.g., a mammalian cellsuch as a Chinese Hamster Ovary (CHO) cell or a COS cell) or aprokaryotic cell. The cell can be derived from a cell line or can be aprimary cell. In one embodiment, the cell is not a transformed cell.

The secreted polypeptide can be an antibody, e.g., a humanized antibody.

The compound can be labeled, e.g., fluorescently labeled. The compoundcan be an antibody, e.g., a fluorescently labeled antibody.

In one embodiment, the binding of the antibody to the secretedpolypeptide on the surface of the cell is detected by flow cytometry.The cell can optionally be selected by fluorescence activated cellsorting.

In another aspect, the invention features a method of selecting a cell,the method including: providing a cell population containing a pluralityof cells genetically engineered to contain a nucleic acid encoding asecreted polypeptide; contacting the cell population with a compoundthat specifically binds to the secreted polypeptide; and selecting acell on the surface of which the compound is bound.

The cell can be, for example, a eukaryotic cell (e.g., a mammalian cellsuch as a Chinese Hamster Ovary (CHO) cell or a COS cell) or aprokaryotic cell. The cell can be derived from a cell line or can be aprimary cell. In one embodiment, the cell is not a transformed cell. Inanother embodiment, the cell is not a B cell or a cell formed by fusionof a B cell and another cell.

The secreted polypeptide can be an antibody, e.g., a humanized antibody.

The compound can be labeled, e.g., fluorescently labeled. The compoundcan be an antibody, e.g., a fluorescently labeled antibody.

In one embodiment, the binding of the antibody to the secretedpolypeptide on the surface of the cell is detected by flow cytometry.The cell can optionally be selected by fluorescence activated cellsorting.

The cell can be selected together with a plurality of cells in the cellpopulation displaying the compound bound to the secreted polypeptide onthe surface of the plurality of cells. The plurality of cells canoptionally contain, e.g., at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, ormore of the cells in the cell population. The plurality of cells canoptionally contain no more than, e.g., at least 1%, 5%, 10%, 20%, 30%,40%, or 50% of the cells in the cell population.

The cell can be deposited in a vessel containing no cells in addition tothe cell.

The methods described herein allow for the simple, fast, and directdetection of secreted polypeptides on the cell surface, optionallyfollowed by cell sorting. High-speed cell sorters can sort hundreds ofmillions of cells with exceptional accuracy, greatly enriching highproducer populations.

An advantage of the invention is that, by using the presence of asecreted polypeptide on the surface of a cell to guide cell selection,the methods can greatly facilitate the process of selecting cellsproducing a given secreted polypeptide. For example, the methods of theinvention can reduce the necessity for carrying out extensive laborintensive and costly assays to detect a polypeptide secreted into cellculture media. In addition, the methods of the invention can reduce thenumber of individual clones that are analyzed during a cell selectionprocess to identify a high producing cell line.

Another advantage of the methods of the invention is that they can beused to comprehensively survey an entire target cell population, sincepotentially all cells present in a transfected or amplified cellpopulation can be examined for the production of a secreted polypeptide.Methods that rely, for example, on cloning do not provide for the directdetection of relative amounts of a polypeptide secreted by all cells ina population. The methods of the invention permit the direct analysis ofa large number of cells and the determination of their relativeexpression levels for a given secreted polypeptide.

Another advantage of the methods of the invention is that, up to thepoint of cloning (if cloning is desired), all cells in a target cellpopulation (e.g., cells transfected with a nucleic acid encoding asecreted polypeptide) can be handled in a single batch. As relativelylittle handling of the cells of target cell population is required, theproduction of multiple cell lines is therefore facilitated. Immunoassaylabor and expense can also be greatly reduced, as initial screeningsteps can be performed by using a flow cytometer.

Another advantage of the invention is that the methods directly detectthe production of a given secreted polypeptide. Methods that insteadrely on the detection of a surrogate marker such as a selectable markeror reporter protein can provide good measures of transcription of anucleic acid encoding a secreted polypeptide, but do not necessarilyprovide a good measure of secretion of the secreted polypeptide. Forexample, increased transcription of a nucleic acid does not necessarilycorrelate with increased translation and secretion of the encodedpolypeptide. The methods of the invention allow for the direct selectionof a cell that possesses the proper cellular machinery and conditionsthat lead to high level production of the secreted polypeptide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the exemplary methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentapplication, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a histogram depicting untransfected CHO cells stained with anRPE labeled goat anti-human antibody.

FIG. 1B is a histogram depicting CHO cells transfected with pXLTBR.9 andstained with an RPE labeled goat anti-human antibody.

FIG. 2A is a histogram depicting pXLTBR.9-transfected CHO cells,following one round of sorting, stained with an RPE labeled goatanti-human antibody.

FIG. 2B is a histogram depicting pXLTBR.9-transfected CHO cells,following two rounds of sorting, stained with an RPE labeled goatanti-human antibody.

FIG. 2C is a histogram depicting pXLTBR.9-transfected CHO cells,following three rounds of sorting, stained with an RPE labeled goatanti-human antibody.

FIG. 3 is a histogram depicting CHO cells transfected with plasmidsencoding the AQC2 mAb and stained with an RPE labeled goat anti-humanantibody, before cell sorting (left) and after cell sorting (right).

DETAILED DESCRIPTION

The present invention provides methods for detecting a secretedpolypeptide on the surface of a cell that produces the polypeptide. Thedetection of a secreted polypeptide on the surface of cells can be usedto select cells based upon the presence or amount of a given secretedpolypeptide produced by the cells.

The screening methods described herein (e.g., screening transfected celllines for cells that are relatively high producers of a heterologouspolypeptide) detect a secreted polypeptide that is transientlyassociated with the plasma membrane during protein secretion. As such,the secreted polypeptide can be labeled with a compound, e.g., afluorescent reagent such as a protein-specific antibody. As described inthe accompanying Examples, fluorescence intensity of labeled secretedpolypeptides on the cell surface was used as the predominant criteriafor the selection of clones and resulted in the selection of cloneshaving relatively high specific productivity of the secretedpolypeptide.

The methods described herein provide for the simple and direct detectionof secreted polypeptides on the cell surface, optionally followed bycell sorting. High-speed cell sorters can sort hundreds of millions ofcells with exceptional accuracy, greatly enriching high producerpopulations, and can deposit one cell per well into plates such as 96well plates. As described in the accompanying Examples, three rounds ofre-iterative sorting followed by single cell seeding was found to resultin clones with specific productivities 20 times higher than the unsortedtransfected cell population. In addition, the selection of clones bycell sorting followed by methotrexate amplification resulted in agreater than 100 fold enrichment in specific productivity.

Secreted Polypeptides

The invention encompasses methods of identifying and selecting cellsexpressing a secreted polypeptide. The secreted polypeptide can be anaturally occurring or a non-naturally occurring protein. The secretedpolypeptide can be produced naturally by a cell (e.g., without anygenetic manipulation of the cell), can be encoded by a heterologousnucleic acid introduced into a cell, or can be produced by a cellfollowing the insertion or activation of sequences that regulateexpression of a gene encoding the secreted polypeptide.

Any polypeptide that is secreted from a cell can be used in the methodsdescribed herein. For example, secreted polypeptides such as cytokines,lymphokines, and/or growth factors can be produced, and cells producingsuch polypeptides can be selected according to the methods describedherein. Examples of such secreted polypeptides include, but are notlimited to, Erythropoietin, Interleukin 1-Alpha, Interleukin 1-Beta,Interleukin-2, Interleukin-3, Interleukin-4, Interleukin-5,Interleukin-6, Interleukin-7, Interleukin-8, Interleukin-9,Interleukin-10, Interleukin-11, Interleukin-12, Interleukin-13,Interleukin-14, Interleukin-15, Lymphotactin, Lymphotoxin Alpha,Monocyte Chemoattractant Protein-1, Monocyte Chemoattractant Protein-2,Monocyte Chemoattractant Protein-3, Megapoietin, Oncostatin M, SteelFactor, Thrombopoietin, Vascular Endothelial Cell Growth Factor, BoneMorphogenetic Proteins, Interleukin-1 Receptor Antagonist,Granulocyte-Colony Stimulating Factor, Leukemia Inhibitory Factor,Granulocyte-Macrophage Colony-Stimulating Factor, MacrophageColony-Stimulating Factor, Interferon Gamma, Interferon Beta, FibroblastGrowth Factor, Tumor Necrosis Factor Alpha, Tumor Necrosis Factor Beta,Transforming Growth Factor Alpha, Gonadotropin, Nerve Growth Factor,Platelet-Derived Growth Factor, Macrophage Inflammatory Protein 1 Alpha,Macrophage Inflammatory Protein 1 Beta, and Fas Ligand. Cells producinga non-naturally occurring, secreted variant of any the abovepolypeptides can also be identified and selected according to themethods described herein.

In addition to the secreted polypeptides described above, the methodsdescribed herein can also be used to produce a fusion protein thatcontains all or a portion of a given protein fused to a sequence ofamino acids that direct secretion of the fusion protein from a cell. Insome cases, such fusion proteins can allow for the secretion of apolypeptide sequence that is not typically secreted from a cell. Forexample, all or a portion of a protein (e.g., a membrane associatedprotein such as a receptor or an intracellular protein) can be fused toa portion of an immunoglobulin molecule (e.g., to the hinge region andconstant region CH2 and CH3 domains of a human IgG1 heavy chain). Inaddition, all or a portion of a protein can be fused to a heterologoussignal sequence. Examples of proteins that can be fused to a sequencethat directs secretion of the fusion protein include, but are notlimited to, receptors (e.g., Lymphotoxin-Beta receptor), includingreceptors for any of the naturally occurring secreted polypeptidesdescribed herein. In preparing a nucleic acid encoding a fusion protein,a naturally occurring transmembrane segment of a cell surface receptorcan be removed to facilitate secretion of the fusion protein encoded bythe nucleic acid.

The secreted polypeptide can be an antibody or an antigen-bindingfragment of an antibody. The antibody can be directed against anantigen, e.g., a protein antigen such as a soluble polypeptide or a cellsurface receptor. For example, the antibody can be directed against acell surface receptor involved in immune cell activation (e.g., CD3,CD4, CD8, CD40, or an integrin such as alpha 1 beta 1 integrin), adisease-associated antigen (e.g., a cancer-associated antigen such asHER2 or prostate specific membrane antigen), or an antigen produced by apathogen (e.g., a viral or bacterial antigen). The particular epitopebound by the antibody can be formed by amino acids, carbohydrates (e.g.,sugars), inorganic moieties (e.g., phosphates), or combinations thereof.Such epitopes can be found in N- or O-linked glycoproteins,proteoglycans, and phosphorylated proteins.

The term “antibody” refers to an immunoglobulin molecule or anantigen-binding portion thereof. As used herein, the term “antibody”refers to a protein containing at least one, for example two, heavychain variable regions (“VH”), and at least one, for example two, lightchain variable regions (“VL”). The VH and VL regions can be furthersubdivided into regions of hypervariability, termed “complementaritydetermining regions” (“CDR”), interspersed with regions that are moreconserved, termed “framework regions” (FR). The antibody can furtherinclude a heavy and light chain constant region, to thereby form a heavyand light immunoglobulin chain, respectively. In one embodiment, theantibody is a tetramer of two heavy immunoglobulin chains and two lightimmunoglobulin chains, wherein the heavy and light immunoglobulin chainsare inter-connected by, e.g., disulfide bonds. The heavy chain constantregion contains three domains, CH1, CH2, and CH3. The light chainconstant region contains one domain, CL. The variable region of theheavy and light chains contains a binding domain that interacts with anantigen.

The secreted polypeptide can be a fully human antibody (e.g., anantibody made in a mouse genetically engineered to produce an antibodyfrom a human immunoglobulin sequence), or a non-human antibody, e.g., arodent (mouse or rat), goat, or primate (e.g., monkey) antibody.

An antibody can be one in which the variable region, or a portionthereof, e.g., the CDRs, are generated in a non-human organism, e.g., arat or mouse. Chimeric, CDR-grafted, or humanized antibodies can be usedas a secreted polypeptide in the methods described herein.

An antibody can be humanized by methods known in the art. For example,humanized antibodies can be generated by replacing sequences of the Fvvariable region which are not directly involved in antigen binding withequivalent sequences from human Fv variable regions. General methods forgenerating humanized antibodies are described by, e.g., Morrison (1985)Science 229:1202-1207.

Nucleic Acids Encoding Secreted Polypeptides

The invention encompasses methods of identifying and selecting cellsexpressing a secreted polypeptide. In some embodiments, the secretedpolypeptide is encoded by a heterologous nucleic acid introduced into acell or is produced by a cell following the insertion or activation ofsequences that regulate expression of a gene encoding the secretedpolypeptide.

Any method for introducing a nucleic acid into a cell can be used toproduce a secreted polypeptide encoded by a heterologous nucleic acid.The nucleic acid can be naked or associated or complexed with a deliveryvehicle. For a description of the use of naked DNA, see e.g., U.S. Pat.No. 5,693,622.

A nucleic acid can be introduced into a cell by a transfection methodsuch as calcium phosphate transfection, transfection using DEAE-Dextran,transfection by electroporation, or transfection using cationic lipidreagents. Suitable transfection methods are described in, e.g., CurrentProtocols in Molecular Biology (1999) John Wiley & Sons, Inc.

Nucleic acids can be delivered to a cell using delivery vehicles, suchas lipids, depot systems, hydrogel networks, particulates, liposomes,ISCOMS, microspheres or nanospheres, microcapsules, microparticles, goldparticles, virus-like particles, nanoparticles, polymers, condensingagents, polysaccharides, polyamino acids, dendrimers, saponins,adsorption enhancing materials, colloidal suspensions, dispersions,powders, or fatty acids.

Viral particles can also be used, e.g., retroviruses, adenovirus,adeno-associated virus, pox viruses, SV40 virus, alpha virus,lentivirus, or herpes viruses, to introduce the heterologous nucleicacid into a cell.

Microparticles or nanoparticles can be used as vehicles for deliveringnucleic acids into a cell. Microparticles can contain macromoleculesembedded in a polymeric matrix or enclosed in a shell of polymer.Microparticles act to maintain the integrity of the macromolecule, e.g.,by maintaining the DNA in a nondegraded state.

Nucleic acid constructs encoding a secreted polypeptide can optionallyinclude a nucleotide sequence encoding a selectable marker or a reporterprotein. In some cases, a nucleotide sequence encoding a selectablemarker or a reporter protein is contained in a second nucleic acidconstruct that is co-introduced into a cell with the nucleic acidconstruct encoding the secreted polypeptide. The selectable marker orreporter protein can provide an additional mechanism, in addition to thescreening methods described herein, for identifying cells containing anucleic acid encoding the secreted polypeptide. Selectable markersinclude, for example, proteins that confer resistance to neomycin,kanamycin, hygromycin, or methotrexate. Reporter proteins include, forexample, beta galactosidase, luciferase, and fluorescent proteins suchas green fluorescent protein. The detection and selection methodsdescribed herein can be carried out in the presence or in the absence ofa selectable marker or a reporter protein.

Selection of a Cell Producing a Secreted Polypeptide

A cell producing a secreted polypeptide can be identified by contactingthe cell with a compound that specifically binds to the secretedpolypeptide and detecting the binding of the compound to the secretedpolypeptide on the surface of the cell. In addition, the cell can beselected from other cells based upon the presence or amount of thecompound bound to the secreted polypeptide on the surface of the cell.“Selecting” a cell includes isolating a single cell into a vesselcontaining only that cell (e.g., single cell sorting for the cloning acell), as well as isolating the cell together with a plurality of cellsbased upon the cells' similar characteristics with respect to thebinding of the compound to the secreted polypeptide on the surface ofthe cells.

A cell can be selected from other cells in a cell population by the useof flow cytometry and cell sorting techniques. In flow cytometry,measurements of cells are made as the cells flow in single file in afluid stream past optical and/or electronic sensors. Flow cytometerstypically use lasers as light sources and measure light scattered bycells, which provides information about their size and internalstructure, and fluorescence in several spectral regions emitted by dyesor labeled probes or reagents that bind specifically andstoichiometrically to cellular constituents such as antigens. Flowsorting allows cells with preselected characteristics to be divertedfrom the stream and collected for further analysis. The optics of a flowcytometer are similar to those of a fluorescence microscope. Flowcytometry and cell sorting are described in detail in, e.g.,Darzynkiewicz et al. (2000) Flow Cytometry, 3rd Edition, San Diego,Academic Press, 2000; and Givan (2001) Flow Cytometry: First Principles,2nd Edition, New York, Wiley-Liss.

For flow cytometry and cell sorting, the compound that specificallybinds to the secreted polypeptide can be a protein such as an antibody.The antibody can have a label, e.g., a fluorescent label, attached toit. Alternatively, a secondary compound (e.g., a secondary antibody) canbe used that specifically binds to a primary antibody, wherein thesecondary compound either contains a label or is bound by anothercompound that contains a label. For example, an antibody that binds tothe secreted polypeptide can be labeled, e.g., biotinylated, and thencontacted to the secreted polypeptide. The antibody-secreted polypeptidecomplex can be detected, e.g., with avidin coupled to a fluorescentlabel.

Cells can be subjected to one ore more rounds of sorting according tothe methods described herein. Multiple rounds of sorting can be used toenrich for cells producing particularly high levels of the secretedpolypeptide. Cells can be cultured between rounds of cell sorting, orcells can be re-sorted without any culture period between the sortingprocedures. Cells can optionally be sorted based upon their expressionof two or more different secreted polypeptide or a secreted polypeptideand a reporter protein. Additional parameters including but not limitedto cell size, cell viability, or the expression of other cell surfacemarkers can also be used in the sorting procedure.

In addition to flow cytometry and cell sorting, cells can be selected bya variety of techniques that allow for the selection of cells having acompound specifically bound to a secreted polypeptide on the surface ofthe cell. Examples of such selection methods include magnetic separationtechniques (e.g., using magnetically labeled compounds such asantibodies that are specifically attracted to magnetic beads) or panningtechniques. For a description of magnetic separation and panningtechniques, see, e.g., Murphy et al. (1992) J. Cell Sci. 1992102:789-98.

In some emobidements, the methods described herein entail detecting thebinding of the compound to the secreted polypeptide on the surface ofthe cell without adding a substance to the cell that encapsulates thecell (e.g., forms a matrix around the cell) and/or immobilizes thesecreted polypeptide near the cell. For example, buffers used forcontacting a compound to a cell and washing unbound compound from thecell can be standard buffers used for flow cytometry and cell sorting(e.g., phosphate buffered saline, optionally including fetal calfserum).

The cells to be detected and/or selected according to the methodsdescribed herein can be maintained in a temperature range ofapproximately 4° C.-10° C. (e.g., about 4° C.) while the cells arecontacted with a compound that binds to the secreted polypeptide as wellas during associated incubation and cell washing periods. The handlingof the cells at a relatively low temperature may facilitate theirretention of the secreted polypeptide on the surface of the cell and thesubsequent detection of the cell by the specific binding of a compound.

Detection and Purification of Secreted Polypeptides

In addition to the cell-associated screening methods described herein, asecreted polypeptide can be detected in tissue culture media followingthe secretion of the polypeptide from a given cell. Such methods can beused to quantitate the amount of secreted polypeptide produced by agiven cell. For example, an aliquot of tissue culture medium from a cellculture containing a cell sorted as described herein can be used todetermine the amount of a given secreted polypeptide contained therein.Such measurements can be used to verify that a cell selected accordingto a method described herein is secreting the secreted polypeptide or issecreting a defined concentration of the secreted polypeptide. Methodsfor the detection of the secreted polypeptide include, but are notlimited to, enzyme linked immunosorbent assay (ELISA),immunoprecipitation, immunofluorescence, enzyme immunoassay (EIA),radioimmunoassay (RIA), and Western blot analysis. Biological assays canalso be carried out to determine the bioactivity of the secretedpolypeptide. The nature of the biological assay can vary according tothe biological function of the secreted polypeptide.

The secreted polypeptide can optionally be purified from tissue culturemedium containing cells that produce the secreted polypeptide.Purification can be accomplished by contacting the culture medium withan affinity agent, e.g., an antibody, that specifically binds to thesecreted polypeptide. The secreted polypeptide can optionally bepurified to homogeneity.

This invention is further illustrated by the following example thatshould not be construed as limiting the scope of the invention.

EXAMPLES Example 1 Direct, Product-Specific Staining of SecretedRecombinant Proteins at the Plasma Membrane with Fluorescently LabeledAntibodies

CHO cells were transfected with the plasmid vectors pAND162 and pAND160,respectively encoding the light and heavy chains of a humanizedmonoclonal antibody to alpha 1 beta 1 integrin (AQC2 mAb). PlasmidpAND162 encodes a neomycin resistance selectable marker, and plasmidpAND160 encodes wild type DHFR. Both plasmids use the CMVintermediate-early promoter, which extends from a restriction siteapproximately 500 bp upstream of the TATA box to a polylinker near theinitiation codon of the native CMV intermediate early gene. The promoterregion includes splice donor and acceptor sites in the 5′ untranslatedregion. The polyadenylation site is derived from human growth hormonevariant sequence.

DHFR deficient DG44 CHO host cells were maintained as spinner culturesin serum free medium containing nucleosides. Transfections were carriedout by electroporation. Transfected cell lines were grown in alpha minusMEM supplemented with 10% dialyzed fetal bovine serum (FBS) (Hyclone,Logan, Utah) and 2 mM Glutamine (Life Technologies, Grand Island, N.Y.).Following electroporation, the cells were cultured in 6-well tissueculture dishes (Becton Dickinson, Franklin Lakes, N.J.). Three dayspost-infection, 400 μg/ml G418 (Geneticin, Life Technologies, GrandIsland, N.Y.) was added to the medium containing alpha minus MEMsupplemented with 10% dialyzed FBS and 2 mM glutamine. Once cells hadreached about 80% confluence, the wells were pooled and sorted.

CHO cells transfected with the pAND162 and pAND160 plasmids encoding thehumanized AQC2 antibody were labeled with a fluorescently labeledanti-human antibody. Staining of the cells was then viewed using laserconfocal microscopy. The cells were kept on ice until confocal analysis.Fluorescent and differential interference contrast photomicrographs wereacquired on a Leica TCS SP confocal microscope equipped with a red laserdiode and Leica confocal software V2.00 build 0368 (Leica Microsystems,Heidelberg GmbH, Germany). Photomicrographs were taken of cells observedthrough a 40× oil immersion objective. Intense staining of the plasmamembrane of the transfected cells with the anti-human antibody wasdetected. No staining of the plasma membrane of the untransfected DG44CHO host cells was detected. As the detectors used in flow cytometry aremore sensitive than those used in laser confocal microscopy, flowcytometry was also used to examine populations of transfected CHO cellsthat had been labeled with fluorescent reagents directed against thesecreted humanized AQC2 antibody (see Example 3).

Example 2 Generation of High Producing Recombinant CHO Cell Lines in theAbsence of Methotrexate

Plasmid pXLTBR.9 contains a nucleotide sequence encoding wild type DHFRas well as a nucleotide sequence encoding the lymphotoxin-beta receptorfused to human IgG domains, C_(H)2 and C_(H)3 (LTbetaR-Ig) (Browning etal. (1995) J. Immunol. 154:33). pXLTBR.9 uses the CMV intermediate-earlypromoter, as described in Example 1 for pAND162 and pAND160.

DG44 CHO cells were transfected with pXLTBR.9 by electroporationaccording to the methods described in Example 1. The pXLTBR.9transfected cell lines were grown in HYQPF-CHO (Hyclone Laboratories,Logan, Utah), a serum-free medium, or Serum-Free alpha plus MEM medium(alpha plus MEMSF), an enriched alpha plus MEM without FBS.

After selection and outgrowth of the pXLTBR.9 transfectants for morethan 14 days, the cells were pooled and labeled with an RPE labeledF(ab′)₂ fragment of an goat anti-human IgG molecule at 4° C. Thesecells, along with stained negative control CHO cells, were subjected toanalytical flow cytometry prior to a preparative sort. FIG. 1A displaysa histogram of negative control, untransfected CHO cells. The FL-2histogram was derived from the combination of the live cell gate (basedon PI exclusion, top left), and the double discrimination gate (pulsewidth vs FSC, to exclude doublets, top right). R2 represents the sortinggate. FIG. 1B displays a histogram of CHO cells transfected withpXLTBR.9. The sort gate R2 was set to collect the brightest 5% of R-PEpositive cells for all three reiterative sorts. The transfected cells(FIG. 1B) contained populations of cells from which the fluorescenceintensity greatly exceeded that of the negative control (FIG. 1A). Forpreparative sorting of the transfected cells, a gate was set thatencompassed cells within the top 5% of the fluorescence intensity of thecell population. The gated cells were sorted and their cell number wasexpanded by culture under selective conditions and the process wasrepeated two more times.

For LTbetaR-Ig producing cell lines, an analytical scan was performedpost-sorting to evaluate the quality of the sort. The analytical scan aswell as the experimentally determined specific productivity rates (SPR)of LTbetaR-Ig in the pools are displayed in FIGS. 2A-2C. Unsortedtransfected cells had a SPR of approximately 0.5 pg/cell/day (pcd). FIG.2A, an analytical scan of a sample of LTbetaR-Ig-producing cellscollected after a first sort, shows that the sort resulted in apopulation with an increased mean fluorescence intensity and acorresponding increase in specific productivity (the SPR values weredetermined after expansion of the cells in culture). FIG. 2B, ananalytical scan of a sample of LTbetaR-Ig-producing cells collectedafter a second sort, demonstrates a progressive increases in bothfluorescence intensity and specific productivity after reiterativesorting. FIG. 2C is an analytical scan of a sample ofLTbetaR-Ig-producing cells collected after a third sort. The threerounds of sorting of the cells improved the SPR average of the pools byapproximately ten-fold to 5.1 pcd (Table 1). TABLE 1 SpecificProductivity Rate of Unsorted And Consecutively Sorted CHO Pools ofLTbetaR-Ig Expressing Cells Demonstrates That Specific ProductivityIncreases with Re-Iterative Sorting Average SPR LTbetaR-Ig pool (pgcell⁻¹ day⁻¹) ± S.D. Unsorted 0.5 ± 0.0 First sort 3.1 ± 0.1 Second sort2 4.5 ± 0.3 Third sort 3 5.1 ± 0.7

For the SPR assay results depicted in Table 1, 2×10⁵ cells were culturedin a 9.6 cm tissue culture dish in 2 ml of serum-containing media. Thecells and supernatant were harvested after three days in culture. TheSPR assay was conducted in triplicate for each sample.

During the third sort, clones were isolated directly from the cytometerinto 96 well plates. Several weeks later, assayed clones demonstrating arange of specific productivity between 4 and 11.5 pcd were maintained(Table 2). The most productive clone demonstrated a twenty-three-foldenrichment in specific productivity, without the need for methotrexateamplification. Fifty clones assayed by ELISA during this enrichmentprocess. The timeline was approximately nine weeks from transfection toidentification of the best clone. TABLE 2 Specific Productivity Rates ofLTbetaR-Ig Expressing CHO Clones Isolated From Pools Subjected to ThreeRe-Iterative FACS Sorts LTbetaR-Ig clone Average SPR (pg cell⁻¹ day⁻¹) 13.6 ± 0.1 3 5.6 ± 0.6 5 11.5 ± 0.2  8 7.5 ± 0.1 21 9.9 ± 1.4 22 7.5 ±0.7 24 9.3 ± 0.8 25 10.5 ± 1.1 

In the experiments depicted in Table 2, triplicate three-day SPR assayswere conducted in serum-containing media for each sample.

In the experiments described herein, cells were harvested before sortingby Accutase treatment (Innovative Cell Technologies, La Jolla, Calif.)and then maintained at 0-4° C. for all subsequent handling. The cellswere passed through a 70 μm nylon mesh (Becton Dickinson Labware,Franklin Lakes, N.J.), washed twice with cold phosphate buffered saline(PBS) (Life Technologies, Grand Island, N.Y.), and then counted andassessed for viability. The cells were pelleted again by centrifugationfor 5 minutes at 1,000 RPM at 4° C., and resuspended in cold DMEM/BSAcontaining fluorescently labeled antibody.

A minimum of 1×10⁷ cells were stained for the detection of plasmamembrane surface LTbetaR-Ig (or humanized AQC2 mAb in the Example 3)with R-phycoerythrin (RPE) conjugated goat F(ab′)2 anti-human IgG(Jackson Immunoresearch, West Grove, Pa.), at a concentration of 0.2-1ug antibody per 1×10⁵ cells in Dulbecco's Minimal Essential Media (DMEM)(Life Technologies, Grand Island, N.Y.), supplemented with 2% BovineSerum Albumin (13SA) (Sigma Chemical Co, St. Louis, Mo.). After a 15minute incubation on ice, the cells were washed twice with cold PBS, andresuspended in PBS plus 2 ug/ml propidium iodide (PI) (Molecular probes,Eugene, Oreg.). Approximately 5×10⁵ cells were removed for pre-analysisby a FACScan cytometer before sorting. Untransfected DG44 CHO cells wereused as a negative control.

Analytical flow cytometry scans were performed on a FACScan flowcytometer (Becton Dickinson, San Jose, Calif.) equipped with Cellquestv3.0 software and an air-cooled argon laser emitting at 488 nm. The PEemission was detected on Fl-2 and the PI emission was detected on Fl-3using a 585 nm band pass filter.

High speed cell sorts were performed on a Moflo flow cytometer(Cytomation, Fort Collins, Colo.), equipped with Summit 3.0 software andan argon laser emitting at 488 nm for fluorescence excitation. The PEemission was detected on Fl-2, using a 670/40 nm band pass filter, andthe PI emission was detected on Fl-4, using a 670/40 band pass filter.Compensation of PE/PI emission spectrum overlap was accomplished usingCytomation's DSP (Digital Signal Processing) board in Summit. Dead cellswere excluded in a FSC vs. Fl-4 dot plot and doublets were excluded in aPSC Width vs. Area dot plot. PE-labeled signal gating was done on a livecell gated Fl-2 histogram. The sorting gate was the combination of thelive cell gate, the double discrimination gate, and the histogram gateon Fl-2.

LTbetaR-Ig titers were determined from tissue culture supernatant byELISA. Assay plates were coated with an LTbetaR-Ig antibody and boundLTbetaR-Ig was detected by anti-human IgG horseradish peroxidase (HRP)conjugate (Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.).The concentration of LTbetaR-Ig was determined by linear regressionanalysis of the standards.

For each population or clone, 1×10⁵ cells were seeded per well of a6-well tissue culture plate (Corning Inc., Corning, N.Y.), in 2 ml ofgrowth media. Assays were performed in triplicate. The cells wereallowed to grow for 3 days, conditioned media was harvested foranalysis, and the cells removed by Accutase and counted. SpecificLTbetaR-Ig (or AQC2 antibody, as in Example 3) titers werequantitatively determined from media samples by ELISA. The SPR measuredin picograms of specific protein per cell per day (pg cell⁻¹day⁻¹), is afunction of both growth rate and productivity, as represented by thefollowing equations:${SPR} = {\frac{{Total}\quad{protein}\quad{mass}}{{Integral}\quad{cell}\quad{area}\quad({ICA})} = {qP}}$${ICA} = \frac{\left( {{{final}\quad{cell}\quad{number}} - {{initial}\quad{cell}\quad{number}}} \right) \times {days}\quad{in}\quad{culture}}{{LN}\left( {{final}\quad{cell}\quad{{number}/{initial}}\quad{cell}\quad{number}} \right)}$

Example 3 Generation of Methotrexate Amplified Recombinant CHO CellLines

The light and heavy chains of a humanized antibody to alpha 1 beta 1integrin (AQC2 mAb) were expressed in CHO cells (as described inExample 1) from separate plasmids pAND162 and pAND160. Aftertransfection and expansion under DFR and G418 selection, the entiretransfected cell population, having a specific productivity of 0.3 pcd,was labeled with a fluorescent F(ab′)₂ fragment of goat anti-human IgG,and the top 2-5% expressing cells as measured by fluorescence intensitywere collected by cell sorting. After approximately one week ofexpansion, sorted cells were subjected to a second sort. The cells wereexpanded again, then deposited at one cell per well into 96 well platesduring a third sort. As in the case of LTbetaR-Ig (Example 2), sortingresulted in a steady increase in the fluorescence intensity of thelabeled cells as well as the measured specific productivity of bothpools and clones.

For the quantitation of AQC2 mAb by ELISA, assay plates were coated withan AQC2 specific antigen fusion protein. Bound AQC2 mAb was detectedwith donkey anti-human IgG (H+ L) horseradish peroxidase conjugate(Jackson ImmunoResearch, West Grove, Pa.).

Approximately 117 clones were expanded into 24 well plates and screenedfor antibody titer. The top expressing clones were further analyzed in aSPR assay. G418 was removed from the highest ten expressing clones,which were then further amplified in media containing either 100 nM or250 nM methotrexate. Amplified pools were screened for antibody titer.Populations exhibiting a qP equal to or greater than 13.5 pcd weresubjected to high speed cell sorting and autocloning of the upper 2%expressing population to 96 well plates. Two of the topantibody-producing clones, clone 5A and 11B had unamplified qPs of 3.3and 8.0 pcd, respectively (Table 3). When clone 5A was amplified in thepresence of 250 nM methotrexate a pool of specific productivity of 16.6pcd was generated. After fluorescent activated cell sorting and cloning,the best producers from 52 clones assayed exhibited qPs of 41.0 and 32.3pcd. Similarly, for clone 11B, cells amplified only in 100 nMmethotrexate had a specific productivity of 18 pcd and produced clonesof up to 32.5 pcd out of approximately 126 clones screened (Table 3).TABLE 3 Specific Productivity Rates of AQC2 mAb Expressing CHO CellsIsolated from Pools Subjected to Three Re-Iterative FACS Sorts Beforeand After Methotrexate (MTX) Amplification SPR # clones screened (pgcell⁻¹ from amplified nM MTX day⁻¹) pool Unamplified 5A — 3.3 parentclone Amplified pool 5AB 250 16.6 52 Amplified 5AB-17 250 41.0 subclone5AB-52 250 32.3 Unamplified 11B — 8.0 parent clone Amplified pool 11BB250 13.5 Amplified 11BB-46 250 25.4 121 subclone 11BB-67 250 27.311BB-68 250 19.9 11BB-83 250 26.9 Amplified pool 11BA 100 17.9 Amplified11BA-1 100 18.4 126 subclone 11BA-30 100 19.9 11BA-41 100 18.2 11BA-47100 26.6 11BA-50 100 26.1 11BA-118 100 28.1 11BA-100 100 32.5

For the experiments depicted in Table 2, triplicate three-day SPR assayswere conducted in serum-containing media for each sample.

FIG. 3 depicts an analytical scan of unsorted CHO cell transfected withplasmids encoding the AQC2 mAb (left) versus amplified clone 11BA-100(right), demonstrating an increase in both mean fluorescence intensityand specific productivity after methotrexate amplification, sorting, andcloning. The FL-2 histograms were derived by analysis of the PE signalwithin the live cell gate. FIG. 3 shows a thirty-two-fold increase inthe mean fluorescence intensity of the top 100 nM methotrexate amplifiedclone 11BA-100, compared to the initial pool of transfectants (whichalso correlates to the high level product secretion). The 250 nMmethotrexate amplified pool had a qP of 13.5 pcd and produced clones ofup to 27 pcd in a similar size screen (Table 3). The increase influorescence intensity correlates to the significant increase in proteinsecretion. As was seen in the case of the LTbetaR-Ig fusion protein(Example 2), fluorescence intensity was a useful surrogate marker forspecific cellular productivity of a secreted protein.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1. A method of selecting a cell producing a secreted polypeptide, themethod comprising: providing a cell population, wherein the cellpopulation comprises a cell comprising a heterologous nucleic acidencoding a secreted polypeptide; contacting the cell population with acompound that specifically binds to the secreted polypeptide; detectingthe binding of the compound to the secreted polypeptide on the surfaceof the cell; and selecting the cell based upon the presence or amount ofthe compound bound to the secreted polypeptide on the surface of thecell.
 2. The method of claim 1, wherein the cell is not a transformedcell.
 3. The method of claim 1, wherein the cell is a eukaryotic cell.4. The method of claim 3, wherein the cell is a mammalian cell.
 5. Themethod of claim 4, wherein the cell is a Chinese Hamster Ovary (CHO)cell.
 6. The method of claim 1, wherein the cell is not a B cell or acell formed by fusion of a B cell and another cell.
 7. The method ofclaim 1, wherein the secreted polypeptide is an antibody.
 8. The methodof claim 7, wherein the antibody is a humanized antibody.
 9. The methodof claim 1, wherein the compound is labeled.
 10. The method of claim 9,wherein the compound is fluorescently labeled.
 11. The method of claim1, wherein the compound is an antibody.
 12. The method of claim 11,wherein the binding of the antibody to the secreted polypeptide on thesurface of the cell is detected by flow cytometry.
 13. The method ofclaim 12, wherein the cell is selected by fluorescence activated cellsorting.
 14. The method of claim 13, wherein the cell is selectedtogether with a plurality of cells in the cell population displaying thecompound bound to the secreted polypeptide on the surface of theplurality of cells.
 15. The method of claim 14, wherein the plurality ofcells comprises at least 1% of the cell population.
 16. The method ofclaim 15, wherein the plurality of cells comprises at least 5% of thecell population.
 17. The method of claim 13, wherein the cell isdeposited in a vessel containing no cells in addition to the cell. 18.The method of claim 13, further comprising: culturing the selected cellto produce a second cell population that produces the secretedpolypeptide; contacting the second cell population with the antibody;detecting the binding of the antibody to the secreted polypeptide on thesurface of a cell in the second cell population; and selecting the cellin the second cell population by fluorescence activated cell sortingbased upon the presence or amount of the antibody bound to the secretedpolypeptide on the surface of the cell.
 19. The method of claim 13,wherein the contacting of the cell population with the antibody iscarried out between 4° C. and 10° C.
 20. The method of claim 19, whereinthe contacting of the cell population with the antibody is carried outat about 4° C.
 21. The method of claim 1, further comprising: culturingthe selected cell in culture medium under conditions that allow forsecretion of the secreted polypeptide into the culture medium; andpurifying the secreted polypeptide from the culture medium.
 22. A methodof generating a cell producing a secreted polypeptide, the methodcomprising: introducing into a cell a heterologous nucleic acid encodinga secreted polypeptide; culturing the cell under conditions that allowfor synthesis of the secreted polypeptide; contacting the cell with acompound that specifically binds to the secreted polypeptide; detectingexpression of the secreted polypeptide by binding of the compound to thesecreted polypeptide on the surface of the cell; and selecting the cellby fluorescence activated cell sorting.
 23. A method of determining thepresence or amount of a secreted polypeptide produced by a cell, themethod comprising: contacting a cell producing a secreted polypeptidewith a compound that specifically binds to the secreted polypeptide,wherein the cell is not a B cell or a cell formed by the fusion of a Bcell with another cell; and detecting the binding of the compound to thesecreted polypeptide on the surface of the cell, to thereby determinethe presence or amount of the secreted polypeptide produced by the cell.24. The method of claim 23, wherein the cell comprises a heterologousnucleic acid encoding the secreted polypeptide.
 25. A method ofselecting a cell, the method comprising: providing a cell populationcomprising a plurality of cells genetically engineered to contain anucleic acid encoding a secreted polypeptide; contacting the cellpopulation with a compound that specifically binds to the secretedpolypeptide; and selecting a cell on the surface of which the compoundis bound.